K4 is a de novo peptide with antibacterial activity on human pathogens. It has a short sequence (14 amino acids), with a cationic N-terminal moiety and an amphipathic ɑ-helix structure. The present paper demonstrates its activity on Vibrio bacteria in a marine environment. It was found non-toxic on marine organisms including Artemia salina, Dicentrarchus labrax, and Magallana gigas at different developmental stages, but influenced the growth of unicellular organisms like microalgae, depending on the algal strain and on K4 concentration. Furthermore, an original approach coupling liquid chromatography (RP-HPLC) and mass spectrometry (MS/MS) allowed us to monitor the degradation time course of the peptide for the first time in conditions close to a hatchery environment, i.e., in the presence of oyster spat. We detected truncated forms over time, and the full K4 was gradually no longer found in these filter-feeder oysters. Finally, using an automated optical density meter, we monitored the growth of several aquatic bacteria identified as pathogenic on animals. K4 had a bactericidal effect on Aeromonas salmonicida and Vibrio splendidus LGP32 at concentrations below 45 μg mL-1. Our results show that K4 could be an environment-friendly alternative to antibiotics, non-toxic to several marine organisms. The use of K4 would be particularly useful to decrease the bacterial load associated with food intake in the early developmental stages of marine animals reared in hatcheries

The cuttlefish (Sepia officinalis) is a cephalopod mollusk distributed on the western European coast, in the West African Ocean and in the Mediterranean Sea. On the Normandy coast (France), cuttlefish is a target species of professional fishermen, so its reproduction strategy is of particular interest in the context of stock management. Egg-laying, which is coastal, is controlled by several types of regulators among which neuropeptides. The cuttlefish neuropeptidome was recently identified by Zatylny-Gaudin et al. (2016). Among the 38 neuropeptide families identified, some were significantly overexpressed in egg-laying females as compared to mature males. This study is focused on crustacean cardioactive peptides (CCAPs), a highly expressed neuropeptide family strongly suspected of being involved in the control of egg-laying. We investigated the functional and structural characterization and tissue mapping of CCAPs, as well as the expression patterns of their receptors. CCAPs appeared to be involved in oocyte transport through the oviduct and in mechanical secretion of capsular products. Immunocytochemistry revealed that the neuropeptides were localized throughout the central nervous system (CNS) and in the nerve endings of the glands involved in egg-capsule synthesis and secretion, i.e. the oviduct gland and the main nidamental glands. The CCAP receptor was expressed in these glands and in the subesophageal mass of the CNS. Multiple sequence alignments revealed a high level of conservation of CCAP protein precursors in Sepia officinalis and Loligo pealei, two cephalopod decapods. Primary sequences of CCAPs from the two species were fully conserved, and cryptic peptides detected in the nerve endings were also partially conserved, suggesting biological activity that remains unknown for the time being.

No antimicrobial peptide has been identified in cephalopods to date. Annotation of transcriptomes or genomes using basic local alignment Search Tool failed to yield any from sequence identities. Therefore, we searched for antimicrobial sequences in the cuttlefish (Sepia officinalis) database by in silico analysis of a transcriptomic database. Using an original approach based on the analysis of cysteine-free antimicrobial peptides selected from our Antimicrobial Peptide Database (APD3), the online prediction tool of the Collection of Anti-Microbial Peptides (CAMPR3), and a homemade software program, we identified potential antibacterial sequences. Nine peptides less than 25 amino acids long were synthesized. The hydrophobic content of all nine of them ranged from 30 to 70%, and they could form alpha-helices. Three peptides possessed similarities with piscidins, one with BMAP-27, and five were totally new. Their antibacterial activity was evaluated on eight bacteria including the aquatic pathogens Vibrio alginolyticus, Aeromonas salmonicida, or human pathogens such as Salmonella typhimurium, Listeria monocytogenes, or Staphylococcus aureus. Despite the prediction of an antimicrobial potential for eight of the peptides, only two-GR21 and KT19-inhibited more than one bacterial strain with minimal inhibitory concentrations below 25 µM. Some sequences like VA20 and FK19 were hemolytic, while GR21 induced less than 10% of hemolysis on human blood cells at a concentration of 200 µM. GR21 was the only peptide derived from a precursor with a signal peptide, suggesting a real role in cuttlefish immune defense.

Aquaculture production is expected to double by 2030, and demands for aquafeeds and raw materials are expected to increase accordingly. Sustainable growth of aquaculture will require the development of highly nutritive and functional raw materials to efficiently replace fish meal. Enzymatic hydrolysis of marine and aquaculture raw materials could bring new functionalities to finished products. The aim of this study was to determine the zootechnical and transcriptomic performances of protein hydrolysates of different origins (tilapia, shrimp, and a combination of the two) in European seabass (Dicentrarchux labrax) fed a low fish meal diet (5%), for 65 days.

Results

Results were compared to a positive control fed with 20% of fish meal. Growth performances, anterior intestine histological organization and transcriptomic responses were monitored and analyzed. Dietary inclusion of protein hydrolysates in the low fish meal diet restored similar growth performances to those of the positive control. Inclusion of dietary shrimp hydrolysate resulted in larger villi and more goblet cells, even better than the positive control. Transcriptomic analysis of the anterior intestine showed that dietary hydrolysate inclusion restored a pattern of intestinal gene expression very close to the pattern of the positive control. However, as compared to the low fish meal diet and depending on their origin, the different hydrolysates did not modulate metabolic pathways in the same way. Dietary shrimp hydrolysate inclusion modulated more metabolic pathways related to immunity, while nutritional metabolism was more impacted by dietary tilapia hydrolysate. Interestingly, the combination of the two hydrolysates enhanced the benefits of hydrolysate inclusion in diets: more genes and metabolic pathways were regulated by the combined hydrolysates than by each hydrolysate tested independently.

Conclusions

Protein hydrolysates manufactured from aquaculture by-products are promising candidates to help replace fish meal in aquaculture feeds without disrupting animal metabolism and performances.

%B BMC Genomics
%V 19
%G eng
%N 396
%R doi.org/10.1186/s12864-018-4780-0
%0 Book Section
%B Biological Resources of Water
%D 2018
%T Egg-Laying in the cuttlefish Sepia officinalis
%A Céline Zatylny-Gaudin
%A Joël Henry
%B Biological Resources of Water
%G eng
%R DOI: 10.5772/intechopen.71915
%0 Journal Article
%J Scientific Reports
%D 2018
%T Emergence of a cholecystokinin/sulfakinin signalling system in Lophotrochozoa
%A Julie Schwartz
%A Marie-Pierre Dubos
%A Jeremy Pasquier
%A Céline Zatylny-Gaudin
%A Pascal Favrel
%X Chordate gastrin/cholecystokinin (G/CCK) and ecdysozoan sulfakinin (SK) signalling systems represent divergent evolutionary scenarios of a common ancestral signalling system. The present article investigates for the first time the evolution of the CCK/SK signalling system in a member of the Lophotrochozoa, the second clade of protostome animals. We identified two G protein-coupled receptors (GPCR) in the oyster Crassostrea gigas (Mollusca), phylogenetically related to chordate CCK receptors (CCKR) and to ecdysozoan sulfakinin receptors (SKR). These receptors, Cragi-CCKR1 and Cragi-CCKR2, were characterised functionally using a cell-based assay. We identified di- and mono-sulphated forms of oyster Cragi-CCK1 (pEGAWDY(SO3H)DY(SO3H)GLGGGRF-NH2) as the potent endogenous agonists for these receptors. The Cragi-CCK genes were expressed in the visceral ganglia of the nervous system. The Cragi-CCKR1 gene was expressed in a variety of tissues, while Cragi-CCKR2 gene expression was more restricted to nervous tissues. An in vitro bioassay revealed that different forms of Cragi-CCK1 decreased the frequency of the spontaneous contractions of oyster hindgut. Expression analyses in oysters with contrasted nutritional statuses or in the course of their reproductive cycle highlighted the plausible role of Cragi-CCK signalling in the regulation of feeding and its possible involvement in the coordination of nutrition and energy storage in the gonad. This study confirms the early origin of the CCK/SK signalling system from the common bilaterian ancestor and delivers new insights into its structural and functional evolution in the lophotrochozoan lineage.
%B Scientific Reports
%V 8
%P 16424
%8 11/2018
%G eng
%U https://www.nature.com/articles/s41598-018-34700-4
%R doi.org/10.1038/s41598-018-34700-4
%0 Journal Article
%J Fish and shellfish Immunology
%D 2018
%T Identification of a moronecidin-like antimicrobial peptide in the venomous fish Pterois volitans: Functional and structural study of pteroicidin-α.
%A Houyvet, Baptiste
%A Yolande Bouchon-Navaro
%A Bouchon, Claude
%A Goux, Didier
%A Bernay, Benoît
%A Corre, Erwan
%A Céline Zatylny-Gaudin
%X

The present study characterizes for the first time an antimicrobial peptide in lionfish (Pterois volitans), a venomous fish. Using a peptidomic approach, we identified a mature piscidin in lionfish and called it pteroicidin-α. We detected an amidated form (pteroicidin-α- CONH2) and a non-amidated form (pteroicidin-α-COOH), and then performed their functional and structural study. Interestingly, the two peptides displayed different antibacterial and hemolytic activity levels. Pteroicidin-α-CONH2 was bactericidal on human pathogens like Staphylococcus aureus or Escherichia coli, as well as on the fish pathogen Aeromonas salmonicida, while pteroicidin-α-COOH only inhibited their growth. Furthermore, the two peptides induced hemolysis of red blood cells from different vertebrates, namely humans, sea bass and lesser-spotted dogfish. Hemolysis occurred with low concentrations of pteroicidin-α-CONH2, indicating greater toxicity of the amidated form. Circular dichroism analysis showed that both peptides adopted a helical conformation, yet with a greater α-helix content in pteroicidin-α-CONH2. Overall, these results suggest that amidation strongly influences pteroicidin-α by modifying its structure and its physico-chemical characteristics and by increasing its hemolytic activity

Sepia officinalis egg protection is ensured by a complex capsule produced by the female accessory genital glands and the ink bag. Our study is focused on the proteins constituting the main egg case. De novo transcriptomes from female genital glands provided essential databases for protein identification. A proteomic approach in SDS-PAGE coupled with MS unveiled a new egg case protein family: SepECPs, for Sepia officinalis Egg Case Proteins. N-glycosylation was demonstrated by PAS staining SDS-PAGE gels. These glycoproteins are mainly produced in the main nidamental glands. SepECPs share high sequence homology, especially in the signal peptide and the three cysteine-rich domains. SepECPs have a high number of cysteines, with conserved motifs involved in 3D-structure. SDS-PAGE showed that SepECPs could form dimers; this result was confirmed by TEM observations, which also revealed a protein network. This network is similar to the capsule network, and it associates these structural proteins with polysaccharides, melanin and bacteria to form a tight mesh. Its hardness and elasticity provide physical protection to the embryo. In addition, SepECPs also have bacteriostatic antimicrobial activity on GRAM- bacteria. By observing the SepECP / Vibrio aestuarianus complex in SEM, we demonstrated the ability of these proteins to agglomerate bacteria and thus inhibit their growth. These original proteins identified from the outer egg case ensure the survival of the species by providing physical and chemical protection to the embryos released in the environment without any maternal protection.

The female genital apparatus of decapod cephalopods contains a symbiotic accessory nidamental gland (ANG) that harbors bacterial symbionts. Although the ANG bacterial consortium is now well described, the impact of symbiosis on Sepia officinalis innate immunity pathways remains unknown. In silico analysis of the de novo transcriptome of ANG highlighted for the first time the existence of the NF-κB pathway in S. officinalis. Several signaling components were identified, i.e. five Toll-like receptors, eight signaling cascade features, and the immune response target gene iNOS, previously described as being involved in the initiation of bacterial symbiosis in a cephalopod gland. This work provides a first key for studying bacterial symbiosis and its impact on innate immunity in S. officinalis ANG.

Sponges and bacteria have lived together in complex consortia for 700 million years. As filter feeders, sponges prey on bacteria. Nevertheless, some bacteria are associated with sponges in symbiotic relationships. To enable this association, sponges and bacteria are likely to have developed molecular communication systems. These may include molecules such as N-acyl-L-homoserine lactones, produced by Gram-negative bacteria also within sponges. In this study, we examined the role of N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL) on the expression of immune and apoptotic genes of the host sponge Suberites domuncula. This molecule seemed to inhibit the sponge innate immune system through a decrease of the expression of genes coding for proteins sensing the bacterial membrane: a Toll-Like Receptor and a Toll-like Receptor Associated Factor 6 and for an anti-bacterial perforin-like molecule. The expression of the pro-apoptotic caspase-like 3/7 gene decreased as well, whereas the level of mRNA of anti-apoptotic genes Bcl-2 Homolog Proteins did not change. Then, we demonstrated the differential expression of proteins in presence of this 3-oxo-C12-HSL using 3D sponge cell cultures. Proteins involved in the first steps of the endocytosis process were highlighted using the 2D electrophoresis protein separation and the MALDI-TOF/TOF protein characterization: α and β subunits of the lysosomal ATPase, a cognin, cofilins-related proteins and cytoskeleton proteins actin, α tubulin and α actinin. The genetic expression of some of these proteins was subsequently followed. We propose that the 3-oxo-C12-HSL may participate in the tolerance of the sponge apoptotic and immune systems towards the presence of bacteria. Besides, the sponge may sense the 3-oxo-C12-HSL as a molecular evidence of the bacterial presence and/or density in order to regulate the populations of symbiotic bacteria in the sponge. This study is the first report of a bacterial secreted molecule acting on sponge cells and regulating the symbiotic relationship.

Since the initial characterization of the cardioexcitatory peptide FMRFamide in the bivalve mollusk Macrocallista nimbosa, a great number of FMRFamide-like peptides (FLPs) have been identified in mollusks. FLPs were initially isolated and molecularly characterized in model mollusks using biochemical methods. The development of recombinant technologies and, more recently, of genomics has boosted knowledge on their diversity in various mollusk classes. Today, mollusk FLPs represent approximately 75 distinct RFamide peptides that appear to result from the expression of only five genes: the FMRFamide-related peptide gene, the LFRFamide gene, the luqin gene, the neuropeptide F gene, and the cholecystokinin/ sulfakinin gene. FLPs display a complex spatiotemporal pattern of expression in the central and peripheral nervous system.Working as neurotransmitters, neuromodulators, or neurohormones, FLPs are involved in the control of a great variety of biological and physiological processes including cardiovascular regulation, osmoregulation, reproduction, digestion, and feeding behavior. From an evolutionary viewpoint, the major challenge will then logically concern the elucidation of the FLP repertoire of orphan mollusk classes and the way they are functionally related. In this respect, deciphering FLP signaling pathways by characterizing the specific receptors these peptides bind remains another exciting objective.

UNLABELLED: We characterized the proteome of the posterior salivary glands of the cephalopod S. officinalis by combining de novo RNA sequencing and mass spectrometry. In silico analysis of the transcriptome revealed the occurrence of three main categories of proteins: enzymes, immune factors and toxins. Protein identification by SDS-PAGE and MALDI-TOF/TOF confirmed the occurrence of proteins essential to venom-like enzymes: peptidase S1 under four isoforms, phospholipase A2 and two toxins. The first toxin is a cystein rich secreted protein (CRISP), a common toxin found in all venomous animals. The second one is cephalotoxin, which is specific to decabrachia cephalopods. Secretions of the posterior salivary glands are transported to the cephalopodium; they are involved in prey catching but also in gamete storage, fertilization and egg-laying. The paralyzing activity and the antimicrobial effect of saliva suggest a dual role in predation and in immune defense in cuttlefish.

BIOLOGICAL SIGNIFICANCE: The originality of this study lies in the use of a transcriptomic approach (de novo RNA sequencing) coupled to a proteomic approach to get an overview of posterior salivary glands in S. officinalis. In cephalopods, these glands are involved in predation, more precisely in paralyzing preys and digesting them. Our in silico analysis equally reveals a role in immune defense as observed in mammals' saliva. Our study also shows the specificity of cuttlefish venom, with the identification of cephalotoxins, proteins that are not found in octopuses. Finally, we show that cuttlefish saliva is a complex mixture that has antibacterial and crippling properties, but no lethal effect.

Varroa destructor is the main concern related to the gradual decline of honeybees. Nowadays, among the various acaricides used in the control of V. destructor, most presents increasing resistance. An interesting alternative could be the identification of existent molecules as new acaricides with no effect on honeybee health. We have previously constructed the first 3D model of AChE for honeybee. By analyzing data concerning amino acid mutations implicated in the resistance associated to pesticides, it appears that pirimicarb should be a good candidate for varroacide. To check this hypothesis, we characterized the AChE gene of V. destructor. In the same way, we proposed a 3D model for the AChE of V. destructor. Starting from the definition of these two 3D models of AChE in honeybee and varroa, a comparison between the gorges of the active site highlighted some major differences and particularly different shapes. Following this result, docking studies have shown that pirimicarb adopts two distinct positions with the strongest intermolecular interactions with VdAChE. This result was confirmed with in vitro and in vivo data for which a clear inhibition of VdAChE by pirimicarb at 10 μM (contrary to HbAChE) and a 100% mortality of varroa (dose corresponding to the LD50 (contact) for honeybee divided by a factor 100) were observed. These results demonstrate that primicarb could be a new varroacide candidate and reinforce the high relationships between in silico, in vitro, and in vivo data for the design of new selective pesticides.

An RNAseq approach associated to mass spectrometry was conducted to assess the composition, molecular mass distribution and primary sequence of hydrolytic peptides issued from hydrolysates of white shrimp (Litopenaeus vannamei) by-products. High performance size exclusion chromatography (HPSEC) analyses indicated that 69.2% of the 214-nm-absorbing components had apparent molecular masses below 1000 Da, and 88.3% below 2000 Da. OFFGEL-nLC-MALDI-TOF/TOF and nLC-ESI-MS/MS analyses led to the identification of 808 peptides based on the NCBI EST databank (161,397 entries) completed by the new L. vannamei databank (58,508 entries) that we created from the RNAs of tissues used for hydrolysate production. Whereas most of hydrolytic peptides have a MW below 2000 Da, preliminary investigations of antimicrobial properties revealed three antibacterial fractions that demonstrate functional activities. The abundance of small peptides as well as the biological activities detected could imply very interesting applications for shrimp hydrolysate in the field of aquaculture feeding.